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Evidence for a causal link between adaptor protein PDZK1 downregulation and Na⁺/H⁺ exchanger NHE3 dysfunction in human and murine colitis.

Yeruva S, Chodisetti G, Luo M, Chen M, Cinar A, Ludolph L, Lünnemann M, Goldstein J, Singh AK, Riederer B, Bachmann O, Bleich A, Gereke M, Bruder D, Hagen S, He P, Yun C, Seidler U - Pflugers Arch. (2014)

Bottom Line: A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine.PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not.We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.

ABSTRACT
A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

No MeSH data available.


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PDZK1 protein expression and NHE3 activity in Pdzk1+/− mice. a PDZK1 protein expression in the small intestinal BBM of Pdzk1+/+, +/−, and −/− mice was analyzed by Western blotting. b Protein bands were quantified using image J software and normalized against β-actin. PDZK1 protein abundance was more than 50 % reduced in the BBM from Pdzk1+/− compared to +/+ mice. c Acid-activated NHE3 activity in cryptal mouth enterocytes from Pdzk1+/− colonic crypts was significantly lower than in colonocytes from control littermates. 6−8 crypts were measured from each colonic crypt preparations and averaged for each of the five pairs of mice. Bar graphs are represented as mean ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005
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Fig8: PDZK1 protein expression and NHE3 activity in Pdzk1+/− mice. a PDZK1 protein expression in the small intestinal BBM of Pdzk1+/+, +/−, and −/− mice was analyzed by Western blotting. b Protein bands were quantified using image J software and normalized against β-actin. PDZK1 protein abundance was more than 50 % reduced in the BBM from Pdzk1+/− compared to +/+ mice. c Acid-activated NHE3 activity in cryptal mouth enterocytes from Pdzk1+/− colonic crypts was significantly lower than in colonocytes from control littermates. 6−8 crypts were measured from each colonic crypt preparations and averaged for each of the five pairs of mice. Bar graphs are represented as mean ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005

Mentions: Another approach to assess the consequence of reduced (but not absent) levels of PDZK1 expression on NHE3 activity was to study PDZK1 expression and NHE3 functional activity in enterocytes from PDZK1 heterozygotes. Acid-activated NHE3 activity was significantly decreased not only in the Pdzk1−/− colonocytes, as previously reported [9], but also in Pdzk1+/− compared to Pdzk1+/+ colonocytes (Fig. 8c). This demonstrates that a reduction (but not absence) in PDZK1 protein expression, similar to that observed in inflamed colon, compromises colonocyte NHE3 function in a similar fashion to that observed in colonocytes from inflamed murine intestine.Fig. 8


Evidence for a causal link between adaptor protein PDZK1 downregulation and Na⁺/H⁺ exchanger NHE3 dysfunction in human and murine colitis.

Yeruva S, Chodisetti G, Luo M, Chen M, Cinar A, Ludolph L, Lünnemann M, Goldstein J, Singh AK, Riederer B, Bachmann O, Bleich A, Gereke M, Bruder D, Hagen S, He P, Yun C, Seidler U - Pflugers Arch. (2014)

PDZK1 protein expression and NHE3 activity in Pdzk1+/− mice. a PDZK1 protein expression in the small intestinal BBM of Pdzk1+/+, +/−, and −/− mice was analyzed by Western blotting. b Protein bands were quantified using image J software and normalized against β-actin. PDZK1 protein abundance was more than 50 % reduced in the BBM from Pdzk1+/− compared to +/+ mice. c Acid-activated NHE3 activity in cryptal mouth enterocytes from Pdzk1+/− colonic crypts was significantly lower than in colonocytes from control littermates. 6−8 crypts were measured from each colonic crypt preparations and averaged for each of the five pairs of mice. Bar graphs are represented as mean ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005
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Related In: Results  -  Collection

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Fig8: PDZK1 protein expression and NHE3 activity in Pdzk1+/− mice. a PDZK1 protein expression in the small intestinal BBM of Pdzk1+/+, +/−, and −/− mice was analyzed by Western blotting. b Protein bands were quantified using image J software and normalized against β-actin. PDZK1 protein abundance was more than 50 % reduced in the BBM from Pdzk1+/− compared to +/+ mice. c Acid-activated NHE3 activity in cryptal mouth enterocytes from Pdzk1+/− colonic crypts was significantly lower than in colonocytes from control littermates. 6−8 crypts were measured from each colonic crypt preparations and averaged for each of the five pairs of mice. Bar graphs are represented as mean ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005
Mentions: Another approach to assess the consequence of reduced (but not absent) levels of PDZK1 expression on NHE3 activity was to study PDZK1 expression and NHE3 functional activity in enterocytes from PDZK1 heterozygotes. Acid-activated NHE3 activity was significantly decreased not only in the Pdzk1−/− colonocytes, as previously reported [9], but also in Pdzk1+/− compared to Pdzk1+/+ colonocytes (Fig. 8c). This demonstrates that a reduction (but not absence) in PDZK1 protein expression, similar to that observed in inflamed colon, compromises colonocyte NHE3 function in a similar fashion to that observed in colonocytes from inflamed murine intestine.Fig. 8

Bottom Line: A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine.PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not.We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.

ABSTRACT
A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

No MeSH data available.


Related in: MedlinePlus